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Hidden-break diversity in pancrustacean rRNA profiles.

Aitana Casanova Gómez1, Francesc Mesquita-Joanes1, Ferran Palero1

  • 1Cavanilles Institute of Biodiversity and Evolutionary Biology, Universidad de Valencia, Paterna, Spain.

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Summary

The hidden break in 28S ribosomal RNA (rRNA) shows new diversity in Oligostraca and Branchiopoda, with Malacostraca displaying varied profiles linked to expansions near D7a. This highlights the need for rRNA-aware quality control in arthropod sample analysis.

Keywords:
28S rRNABranchiopodaD7aFragment analyzerHidden breakMalacostracaOligostracaPancrustacearRNA secondary structure

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Area of Science:

  • Molecular Biology
  • Genomics
  • Invertebrate Zoology

Background:

  • Many invertebrates possess a hidden break in their 28S ribosomal RNA (rRNA) molecule, splitting it into 28S ribosomal RNA subunit alpha (28Sa) and 28S ribosomal RNA subunit beta (28Sb) fragments.
  • This phenomenon masks the full 28S ribosomal RNA (rRNA) in electrophoretic profiles, potentially biasing standard RNA quality measurements in extracted tissue samples.
  • The diversity of RNA hidden breaks across Pancrustacea, especially within Oligostraca, remains largely uncharacterized.

Purpose of the Study:

  • To investigate the diversity of 28S ribosomal RNA (rRNA) hidden breaks in Oligostraca and compare it with Branchiopoda and Malacostraca.
  • To refine fragment size estimation of 28S ribosomal RNA (rRNA) subunits using high-resolution capillary electrophoresis.
  • To correlate observed RNA profile heterogeneity with specific secondary-structure domains of the 28S ribosomal RNA (rRNA), particularly D3 and D7a.

Main Methods:

  • Sampling of 12 invertebrate species from Branchiopoda, Malacostraca, and Oligostraca near Valencia, Spain.
  • RNA stabilization using DNA/RNA Shield, extraction via Quick-RNA MagBead, and subsequent profiling on an Agilent 5200 Fragment Analyzer.
  • Assignment of RNA peaks using BLAST-inferred gene lengths and analysis of 28S secondary-structure domains (D-regions) with RNAfold.

Main Results:

  • Oligostracans and most branchiopods exhibited a canonical profile with 18S, 28Sa, and 28Sb fragments of similar sizes.
  • Malacostracans displayed diverse profiles with multiple peaks, indicating expansions that alter the relative sizes of 28Sa and 28Sb, notably near the D7a domain.
  • Comparative analysis revealed conserved D3/D7a architecture in Oligostraca/Branchiopoda, contrasting with higher variability and frequent expansions in Malacostraca.

Conclusions:

  • The study extends the known diversity of RNA profiles to include Oligostraca, providing refined fragment-size estimates.
  • Malacostracan RNA profile heterogeneity is linked to expansions, particularly around the D7a domain.
  • Recommendations include implementing rRNA-aware quality control for arthropod samples and further sequencing of understudied lineages to elucidate hidden break mechanisms and phylogenetic distribution.